The long-term goal of this research is to decrease the morbidity and mortality related to diabetic vascular dysfunction and related complications, by developing novel, bioactive therapeutic agents. Hyperglycemia- induced vascular inflammation, leading to monocyte adhesion to vascular endothelial cells (ECs), is the key factor that initiates the pathogenesis of atherosclerosis in diabetic patients. Our studies found that botanical genistein inhibits hyperglycemia-induced human leukocyte-EC interaction, suggesting a potential anti- inflammatory action of genistein in diabetic vascular complications. Leukocyte adhesion to endothelium is mediated through both chemokines and adhesion molecules on ECs, and the expression of these molecules is critically up-regulated by nuclear factor kB (NF-kB). Further analysis showed that genistein suppressed high glucose-induced production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in human aortic ECs (HAECs), two chemokines that are the key factors in the firm adhesion of monocytes to activated ECs. Recent studies in this laboratory found that genistein is an activator of the cAMP signaling system in ECs. Interestingly, our studies showed that dietary intake of genistein increased plasma cAMP but reduced MCP-1 levels in diabetic mice. Our specific aim of this proposal is to determine the molecular mechanism by which genistein suppresses hyperglycemia-induced vascular inflammation. We hypothesized that genistein, by activation of the cAMP/PKA signaling, inhibits high glucose-induced monocyte adhesion to ECs through suppression of NF-kB activity and subsequent inhibition of chemokine and adhesion molecule expression. Specific aim 1 will use HAECs to investigate: 1) whether genistein inhibits hyperglycemia-induced expression of chemokines and adhesion molecules;2) whether genistein inhibits hyperglycemia-induced NF-kB activity;and 3) whether the anti-inflammatory properties of genistein are mediated via the cAMP/PKA pathway. Specific aim 2 will use diabetic mouse models to determine: 1) whether genistein induces cAMP/PKA signaling in vivo;2) whether genistein inhibits chemokine and adhesion molecule production from blood vessels;and 3) whether genistein suppresses monocyte recruitment to vascular ECs. Genistein will be administered through a nutritional supplementation approach to both chemically induced and obese diabetic mice. The protein and mRNA expression will be assessed by Western blot, ELISA or real-time PCR. The adenylate cylase activity, cAMP and PKA activation in ECs will be determined using assay kits. NF-kB activity will be determined with ELISA and reporter gene assay. The role of the cAMP/PKA cascade in the anti-inflammatory action of genistein will be determined by using both pharmacological and molecular intervention approaches. The mechanistic understanding of how genistein protects against diabetes-caused vascular inflammation will facilitate the development of efficacious CAM strategies to protect diabetic patients from vascular complications. PUBLIC HEALTH RELEVANCE: Cardiovascular disease such as atherosclerosis is the main cause of deaths in patients with diabetes mellitus. It is increasingly recognized that vascular inflammation mediated by sustained elevation of blood glucose levels in diabetic patients plays a pivotal role in the pathogenesis of atherosclerosis. Our studies indicate that botanical genistein is a promising agent to protect against chronic high glucose- caused vascular inflammation. The results of this research will potentially lead us to develop novel, natural, and cost-effective agents to prevent and treat atherosclerosis caused by diabetes, a chronic disease that affects over 20 million Americans.